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Development of in Vivo Flux Analysis of Hepatic Glucose Production in Type 2 Diabetes

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Development of in Vivo Flux Analysis of Hepatic Glucose Production in Type 2 Diabetes Gluconeogenesis as a System: Development of in vivo Flux Analysis of Hepatic Glucose Production in Type 2 Diabetes By José Orlando Alemán B.S. Chemical Engineering Cornell University, 2001 SUBMITTED TO THE HARVARD-MIT DIVISION OF HEALTH SCIENCES AND TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MEDICAL ENGINEERING AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2008 © 2008 Massachusetts Institute of Technology All rights reserved Signature of Author_________________________________________________________ Harvard-MIT Division of Health Sciences and Technology September 24, 2007 Certified by________________________________________________________________ Gregory Stephanopoulos, PhD Willard Henry Dow Professor of Chemical Engineering and Biotechnology Massachusetts Institute of Technology Thesis Supervisor Accepted by_______________________________________________________________ Martha L. Gray, Ph.D. Edward Hood Taplin Professor of Medical and Electrical Engineering Co-Director, Harvard-MIT Division of Health Sciences and Technology Gluconeogenesis as a System: Development of in vivo Flux Analysis of Hepatic Glucose Production in Type 2 Diabetes by José O. Alemán Submitted to the Division of Health Sciences and Technology on September 24, 2007 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Engineering ABSTRACT Metabolic diseases are an increasing health concern in the developed world. Type 2 Diabetes, (T2D) affects over 100 million people worldwide and significantly contributes to chronic diseases such as atherosclerosis and kidney failure. This condition is characterized by deregulation of glucose homeostasis through the development of insulin resistance, manifested as increased glucose production in the liver. Hepatic gluconeogenesis provides de novo formation of glucose from three- carbon precursors such as glycerol, lactate, pyruvate and alanine. The upregulation of this pathway underlies the persistent hyperglycemia observed in diabetic patients. We have developed stable isotope tracer methods to reconstruct hepatic glucose production fluxes by infusion of [13C, 2H]- glycerol and mass spectrometry analysis of plasma metabolites. Using this methodology we observe physiologic changes in liver cell lines and primary hepatocyte cultures in the presence of hormones insulin/glucagon and in response to gluconeogenic precursor availability. We put forth the hypothesis that in the presence of glycerol as a gluconeogenic susbtrate, glucose-6-phosphatase has an important role in modulating metabolic flux through upper gluconeogenesis. Infusion of 13 2 simultaneous tracer combinations in vivo including a novel [U- C, H5]-glycerol allow detailed net flux and reversibility reconstruction of upper gluconeogenesis to an unprecedented degree in a single experiment. We deployed the developed methods to probe glucose overproduction in the liver insulin receptor knockout (LIRKO) transgenic model of Type 2 Diabetes, and found unexpected similarities in the metabolic flux profile not observed by genomic, protein or metabolite measurements. Our results underscore the importance of flux measurement as a physiologic parameter akin to gene and protein expression in revealing the metabolic phenotype of cells, tissues and organisms. These methods have the potential to contribute as clinical assays to characterize excess glucose production as well as in drug development for new targets to control hepatic glucose production. Thesis Supervisor: Gregory Stephanopoulos Title: Willard Henry Dow Professor of Biotechnology and Chemical Engineering Page 3 Acknowledgments MD-PhD students traverse a challenging path between the demands of clinical medicine and the pursuit of science needed to propel the medical field forward. I chose my doctoral dissertation project as an opportunity to bridge these two interests given my backgrounds in engineering and biomedicine that could open avenues for new research. Time will tell the success of this endeavor, but I hope our efforts remind you the importance of technology development in establishing novel insight to biological processes related to metabolic disease. I would like to thank my advisor, Gregory Stephanopoulos, for providing the flexibility and latitude to pursue a project bridging the medical and engineering pillars of my combined degree education. His constant optimism will serve as inspiration for my nascent career as a scientist. I would also like to thank my thesis committee members Maria Alexander-Bridges, C. Ronald Kahn, Joanne Kelleher, Isaac Kohane and David Rhoads. Individually and as a group, their advice regarding potential avenues of research and how to improve my existing work was critical to the document presented here. In particular, I would like to thank Ron for opening the doors to his laboratory willingly and permit our study of the LIRKO model. Maria was always an encouraging and understanding presence in discussing experimental results. Joanne Kelleher provides a sorely needed physiology perspective at MIT and was instrumental in establishing what is known in this field experimentally. I thank Zak for serving as thesis committee chair and believing in my potential at such early stages; he introduced me to the Bioinformatics and Integrative Training Genomics Grant during my PhD interview! Finally, David Rhoads heed the call for help as a thesis committee member to provide a longitudinal view of the research in our laboratory. Within the Stephanopoulos Group, many individuals contributed to the development of the work presented here. Matthew Wong taught me how to isolate hepatocytes, how to use mass spectrometry and introduced me to fantasy baseball. Maciek Antoniewicz is the theorist behind the elementary metabolite units that permit the interpretation of mass spectrometry data in the analysis of mammalian metabolic networks. Without his insight and expertise, this thesis would not have been possible. Kudos to Jamey Young for actually reading this thesis and helping me improve its organization and focus. Yasushi Noguchi has been a steady experienced hand in performing all animal experiments presented here. Lalisse Guillen was an undergraduate student through the MIT Summer Research Program who helped characterize flux response to energy modulating drugs. In addition, I would like to thank all Stephanopoulos group past and present members who made the lab an eclectic and fun place to work. When the questions asked extended beyond the realm of my expertise, many collaborators lent a hand to make this work better. The authors would like to acknowledge Laura Vineyard and Linda Griffith for kind donation of rat hepatocytes. Chris Autieri and Katie Madden at the Division of Comparative Medicine at MIT were instrumental in setting up our surgical system for ALZET pump implants. At Joslin, Sudha Biddinger was a nurturing postdoctoral influence and reference to all things LIRKO. In addition, Cullen Taniguchi believed in the potential of our early work enough to trust me with his mice. Between these two individuals, I had guidance on how to navigate being a combined degree student for the short and long-term perspectives. Research is expensive, and I would be remiss not to mention funding sources, which included a NSF Graduate Research Fellowship and the Bioinformatics and Integrative Genomics Training Grant at the Division of Health Sciences and Technology. This research work was supported by National Institutes of Health Bioengineering Research Partnership Grant DK-58533, the NIH Metabolomics Roadmap Initiative DK070291, RO1 DK075850 (to Professor Stephanopoulos) and the Dupont-MIT Alliance. Page 4 Through these past five years, I acquired many friends who willingly gave me their support through thick and thin. The chemical engineering graduate community embraced me as one of their own, and in no small degree this influenced the positive outcome of my qualifying examinations. Bernat Olle remained a treasured Catalan connection to my experiences as a Fulbright scholar in Spain. Kris Wood, Jane Rempel, Gregg Beckham, Ben Wang and many others formed a supportive network. Joe Shuga was an esteemed desk and lab neighbor. At the medical school, Iris Bonilla was my class sidekick who reminded me that there is life after the PhD and Boards. I want to thank my family for understanding my self-imposed exile as part of academic preparation. Having left Puerto Rico over a decade ago to pursue my university studies, they remain a close and constant presence that pushes me forward in times of distress. My parents José and Aixa unconditionally supported my dream of becoming a physician-scientist and the multiple turns of this career path. My sisters Yomaris and Maricelis took flight shortly after me but remain grounded and driven to succeed in their respective fields. I dedicate this work to my grandmother Marina and my grandparents Jaime and Carmen who passed away during my graduate studies. Mil gracias, los quiero mucho y volveré pronto… Lastly, I want to thank my wife Tara for her constant support, company and love during this journey toward the PhD. No words can fully capture the extent of her contributions, but she made it possible for me to wake every day and face the challenges and uncertainty of research with a smile. Page 5 Gluconeogenesis as a System: Development of in vivo Flux Analysis of Hepatic Glucose Production in Type 2 Diabetes Table of Contents Gluconeogenesis as a System: Development
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